This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We describe a rapid and efficient method for the identification of phosphopeptides, which we term mass spectrometric (MS) phosphopeptide fingerprinting. The method involves quantitative comparison of proteolytic peptides from native versus completely dephosphorylated proteins. Dephosphorylation of serine, threonine, and tyrosine residues is achieved by in-gel treatment of the separated proteins with hydrogen fluoride (HF). This chemical dephosphorylation results in enrichment of those unmodified peptides that correspond to previously phosphorylated peptides. Quantitative comparison of the signal-to-noise ratios of peaks in the treated versus untreated samples are used to identify phosphopeptides, which can be confirmed and further studied by tandem mass spectrometry (MS-MS). We have applied this method to identify eight known phosphorylation sites of Xenopus Aurora-A kinase, as well as several novel sites in the Xenopus chromosome passenger complex (CPC). During the course of this work, we also developed and implemented a robust method for calculation of signal to noise ratio in mass spectra. A manuscript describing this work has been published: Efficient identification of phosphorylation by mass spectrometric phosphopeptide fingerprinting. Woo EM, Fenyo D, Kwok BH, Funabiki H, Chait BT. Anal Chem. 2008 80(7):2419-25. This method has been applied to the elucidation of a novel protein that appears to be involved in nuclear reformation after mitosis. A manuscript describing this work is in preparation.